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Universal Primers Used for Species Identification of Foodstuff of Animal Origin: Effects of Oligonucleotide Tails on PCR Amplification and Sequencing Performance

机译:用于动物源食品物种鉴定的通用引物:寡核苷酸尾巴对PCR扩增和测序性能的影响

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摘要

M13 universal non-homologous oligonucleotide tails incorporated into universal primers have been shown to improve amplification and sequencing performance. However, a few protocols use these tails in the field of food inspection. In this study, two types of M13 tails (by Steffens and Messing) were selected to assess their benefits using universal cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene (16SrRNA) primers in standard procedures. The primer characteristics were tested in silico. Then, using 20 DNA samples of edible species (birds, fishes, and mammals), their performance during PCR amplification (band recovery and intensity) and sequencing (sequence recovery, length, and Phred score) was assessed and compared. While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing's tails negatively affected the reaction outputs, while Steffens' tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. This different performance could be related to a destabilization effect of certain tails on primers with unfavorable mismatches on the annealing region. Even though our results cannot be generalized because the tail performances are strictly dependent on laboratory conditions, they show that appropriate tails can improve the overall throughput of the analysis, supporting food traceability.
机译:已显示掺入通用引物中的M13通用非同源寡核苷酸尾巴可改善扩增和测序性能。但是,在食品检查领域,一些协议使用了这些尾巴。在这项研究中,选择了两种类型的M13尾巴(由Steffens和Messing进行研究),以在标准程序中使用通用细胞色素氧化酶亚基I(COI)和16S核糖体RNA基因(16SrRNA)引物来评估它们的益处。底漆特性在计算机上进行了测试。然后,使用20种可食用物种(鸟类,鱼类和哺乳动物)的DNA样品,评估和比较它们在PCR扩增(条带恢复和强度)和测序(序列恢复,长度和Phred得分)过程中的性能。尽管16SrRNA尾部引物和非尾部引物的表现相似,但发现COI引物存在差异。混乱的尾巴会对反应输出产生负面影响,而史蒂芬斯的尾巴则根据单个双向读取序列显着改善了带强度和最终重叠群的长度。这种不同的性能可能与某些尾部对引物的去稳定作用有关,这些引物在退火区具有不利的错配。尽管由于尾部性能严格取决于实验室条件而无法将我们的结果进行概括,但它们表明适当的尾部可以提高分析的总体通量,从而支持食品的可追溯性。

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